Cellular and Molecular Physiology Laboratory and Perinatology Research Laboratory, Division of Obstetrics and Gynecology, School of Medicine, Faculty of Medicine, Pontificia Universidad Católica de Chile, Santiago, Chile.
J Cell Physiol. 2011 Nov;226(11):2916-24. doi: 10.1002/jcp.22635.
Insulin causes endothelium-derived nitric oxide (NO)-dependent vascular relaxation, and increases L-arginine transport via cationic amino acid transporter 1 (hCAT-1) and endothelial NO synthase (eNOS) expression and activity in human umbilical vein endothelium (HUVEC). We studied insulin effect on SLC7A1 gene (hCAT-1) expression and hCAT-transport activity role in insulin-modulated human fetal vascular reactivity. HUVEC were used for L-arginine transport and L-[(3) H]citrulline formation (NOS activity) assays in absence or presence of N-ethylmaleimide (NEM) or L-lysine (L-arginine transport inhibitors). hCAT-1 protein abundance was estimated by Western blot, mRNA quantification by real time PCR, and SLC7A1 promoter activity by Luciferase activity (-1,606 and -650 bp promoter fragments from ATG). Specific protein 1 (Sp1), and total or phosphorylated eNOS protein was determined by Western blot. Sp1 activity (at four sites between -177 and -105 bp from ATG) was assayed by chromatin immunoprecipitation (ChIP) and vascular reactivity in umbilical vein rings. Insulin increased hCATs-L-arginine transport, maximal transport capacity (V(max) /K(m) ), and hCAT-1 expression. NEM and L-lysine blocked L-arginine transport. In addition, it was trans-stimulated (∼7.8-fold) by L-lysine in absence of insulin, but unaltered (~1.4-fold) in presence of insulin. Sp1 nuclear protein abundance and binding to DNA, and SLC7A1 promoter activity was increased by insulin. Insulin increased NO synthesis and caused endothelium-dependent vessel relaxation and reduced U46619-induced contraction, effects blocked by NEM and L-lysine, and dependent on extracellular L-arginine. We suggest that insulin induces human umbilical vein relaxation by increasing HUVEC L-arginine transport via hCATs (likely hCAT-1) most likely requiring Sp1-activated SLC7A1 expression.
胰岛素引起内皮衍生的一氧化氮 (NO) 依赖的血管舒张，并通过阳离子氨基酸转运体 1 (hCAT-1) 和内皮型一氧化氮合酶 (eNOS) 的表达和活性增加人脐静脉内皮细胞 (HUVEC) 中的 L-精氨酸转运。我们研究了胰岛素对 SLC7A1 基因 (hCAT-1) 表达的影响以及 hCAT 转运活性在胰岛素调节人胎儿血管反应性中的作用。在不存在或存在 N-乙基马来酰亚胺 (NEM) 或 L-赖氨酸 (L-精氨酸转运抑制剂) 的情况下，使用 HUVEC 进行 L-精氨酸转运和 L-[(3)H]瓜氨酸形成 (NOS 活性) 测定。通过 Western blot 估计 hCAT-1 蛋白丰度，通过实时 PCR 定量 mRNA，通过荧光素酶活性 (-1,606 和 -650 bp 启动子片段从 ATG) 测定 SLC7A1 启动子活性。通过 Western blot 测定特定蛋白 1 (Sp1)、总或磷酸化 eNOS 蛋白。通过染色质免疫沉淀 (ChIP) 和脐带血管环的血管反应性测定 Sp1 活性 (在 ATG 前的 -177 到 -105 bp 之间的四个位点)。胰岛素增加 hCATs-L-精氨酸转运、最大转运能力 (V(max)/K(m)) 和 hCAT-1 表达。NEM 和 L-赖氨酸阻断 L-精氨酸转运。此外，在没有胰岛素的情况下，它被 L-赖氨酸反式刺激 (∼7.8 倍)，但在有胰岛素的情况下没有改变 (~1.4 倍)。胰岛素增加 Sp1 核蛋白丰度和与 DNA 的结合，以及 SLC7A1 启动子活性。胰岛素增加一氧化氮合成并引起内皮依赖性血管舒张，并减少 U46619 引起的收缩，这些作用被 NEM 和 L-赖氨酸阻断，并依赖于细胞外 L-精氨酸。我们认为，胰岛素通过增加 hCATs (可能是 hCAT-1) 介导的 HUVEC L-精氨酸转运来诱导人脐静脉舒张，这可能需要 Sp1 激活的 SLC7A1 表达。
